THP-1 human monocytic cells were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA) and maintained in RPMI-1640 medium (Thermo Fisher Scientific Inc., Waltham, MA, USA) and human umbilical vein endothelial cells (HUVECs) were purchased from ATCC and maintained in Dulbecco's modified Eagle's medium(DMEM)/F12 (Thermo Fisher Scientific Inc.). The two media were supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen; Thermo Fisher Scientific Inc.), 100 units/ml penicillin and 100 µg/ml streptomycin in a humidified atmosphere of 5% CO₂ at 37°C. All cells were confirmed to be free of mycoplasma contamination.

The exosome suspension was added to an equal volume of 4% paraformaldehyde (Nacalai Tesque, Inc., Kyoto, Japan), and the mixture was applied to a Formvar/Carbon film-coated transmission electron microscope (TEM) grid (Alliance Biosystems, Osaka, Japan). Then, the sample was fixed by incubation with 1% glutaraldehyde for 5 min, washed with PBS, and incubated with 1% uranyl acetate for 5 min at 4°C. The sample was observed under a TEM (Hitachi H7650; Hitachi, Ltd., Tokyo, Japan).

To isolate the exosomes, THP-1 exosomes were first treated with 50 µg/ml oxLDL (Shanghai Leuven Biotechnology Co., Ltd., Shanghai, China) for 48 h. The supernatant was subsequently collected and centrifuged at 1,000 × g for 10 min, then 3,000 × g for 30 min at 4°C to remove cell components and fragments. A total exosome isolation kit (Thermo Fisher Scientific Inc.) was then added to the sample overnight at 4°C, prior to centrifugation at 10,000 × g for 1 h at 4°C. Exosomes were re-suspended in PBS and stored at −80°C. The exosome concentration was detected using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology, Hangzhou, China). Exosomes were then co-cultured with 10⁵ HUVECs in 50 ng/ml DMEM for 24 h at 37°C.

Western blot analysis was performed to analyse the expression of the exosomal marker cluster of differentiation (CD)63. Cells were lysed with radioimmunoprecipitation assay buffer (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1% Triton X-100 and 0.5% sodium deoxycholate) and blocked with the complete mini protease inhibitor cocktail 30 min at 4°C (Roche Diagnostics GmbH, Mannheim, Germany). The protein concentration was detected using a bicinchoninic acid protein kit (Beyotime Institute of Biotechnology). A total of 20–30 µg lysate samples were subsequently separated on 8–12% SDS-PAGE gel and transferred onto polyvinylidene membranes. The membranes were incubated with the following primary antibodies overnight at 4°C: rabbit anti-human CD63 (cat. no. ab59479, 1:1,000; Abcam, Cambridge, UK), rabbit anti-human CD9 (cat. no. ab92726, 1:1,000; Abcam), mouse anti-human CD81 (cat. no. H00000975-B01P, 1:500; Novus Biologicals, LLC, Littleton, CO, USA) and mouse anti-Actin (cat. no. CP01-1EA, 1:10,000; Merck KGaA, Darmstadt, Germany). Membranes were then incubated with the following horse radish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (cat. no. 7074; 1:10,000; CST Biological Reagents Co., Ltd., Shanghai, China) and HRP-anti-mouse antibody (cat. no. 7076; 1:10,000; CST Biological Reagents Co., Ltd.) at room temperature for 1 h. The antibodies were detected using an enhanced chemiluminescence kit (cat. no. PI32209; Pierce; Thermo Fisher Scientific Inc.).

Purified exosomes stained with PHK67 (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) according to the manufacture's protocols. Briefly, exosomes resuspended in a buffer provided in the kits were mixed with the PKH dyes and were incubated for 5 min at room temperature. Next, the samples were added to PBS supplemented with 5% bovine serum albumin and were ultracentrifuged at 100,000 g for 1 h at 4°C to remove free dyes. Then, the stained exosomes were co-cultured with HUVECs which were then fixed with 4% paraformaldehyde at 25°C for 20 min.

Macrophages were treated with or without 5 µg/ml GW4869 (dissolved in DMSO; MedChem Express, USA) for 24 h at 37°C to inhibit exosome secretion.

The CCK8 assay was conducted according to the manufacturer's protocol (CK04; Dojindo Molecular Technologies, Inc., Kumamoto, Japan). Exosome-treated cells were plated at equal cell density (2,000 cells/100 µl well) in 96-well plates with 300 µg/ml cetuximab (Merck KGaA) for continuous detection over a 5-day period. At the beginning of the second day, the culture was terminated with the addition of 10 µl CCK8 (5 mg/ml) to the original culture medium. Following 2 h, plates were measured using a microplate reader (Biotek Elx800, Biotek Instruments, Inc., Winooski, VT, USA). Cell proliferation was measured using optical density at 450 nm.

The exosome-treated HUVECs (5×10⁴/96 well plate) were re-suspended in the conditioned medium and seeded into growth factor-reduced Matrigel (356234; BD Biosciences, Franklin Lakes, NJ, USA). Following incubation for 6 h images of the tubes formed were captured under an inverted microscope at ×100 magnification. The length of tubes was measured using ImageJ software version 1.47 (National Institutes of Health, Bethesda, MD, USA).

Statistical analysis was performed using the Student's t-test to compare between groups. Data were presented as mean ± standard error of the mean. A Tukey test was conducted for multiple comparisons in conjunction with one way analysis of variance. P<0.05 was considered to indicate a statistically significant difference. SPSS version 13.0 was used for analysis (SPSS, Inc., Chicago, IL, USA).

To investigate the function of oxLDL-stimulated macrophage-derived exosomes in atherosclerosis, exosomes were isolated from oxLDL-treated or untreated THP-1 cells. The morphology of exosomes collected was observed with scanning electron microscopy. The diameters of the exosomes ranged from 30–120 nm (Fig. 1A). Western blot analysis revealed that exosomes were enriched with the exosomal markers CD9, CD63 and CD81 whereas actin was enriched in the cellular lysate (Fig. 1B), indicating an effective isolation of exosomes.

PHK67 labeled exosomes were co-cultured with HUVECs. Following 24 h, co-cultured HUVECs were fixed and observed by fluorescent microscopy. Exosomes were observed inside the co-cultured HUVECs (Fig. 2), verifying that endothelial cells endocytosed the exosomes derived from oxLDL-stimulated macrophages.

The growth and tube formation abilities of HUVECs co-cultured with exosomes isolated from oxLDL-treated or untreated macrophages were analysed to explore the effects of oxLDL-stimulated macrophage-derived exosomes on endothelial cells. CCK8 assay results revealed that oxLDL-stimulated macrophage-derived exosomes decreased the cell growth ability of HUVECs significantly, compared with exosomes isolated from untreated macrophages (Fig. 3A). Furthermore, tube formation assay results demonstrated that incubation with oxLDL-stimulated macrophage-derived exosomes reduced the tube formation ability of HUVECs compared with to the untreated control (Fig. 3B). HUVECs were also treated with 50 ng/ml oxLDL instead of exosomes. The CCK8 assay and tube formation assay results revealed that 50 ng/ml oxLDL treatment did not suppress the growth and tube formation (Fig. 3), indicating that specifically the exosomes derived from oxLDL-stimulated macrophages reduced the growth and tube formation abilities of endothelial cells.

To further verify the effects of oxLDL-stimulated macrophage-derived exosomes on endothelial cells, exosome generation was blocked with exosomal release inhibitors GW4869 (20,21) (Fig. 4A). CCK8 assay results revealed that exosomes collected from oxLDL-stimulated macrophages treated with the GW4869 inhibitor recovered the cell growth ability of HUVECs compared with the dimethyl sulfoxide (DMSO) control (Fig. 4B). The tube formation assay results also indicated that the exosomes collected from the oxLDL-stimulated macrophages treated with the GW4869 inhibitor regained tube formation ability compared with the DMSO control (Fig. 4C). These results indicated that the blockade of exosomal secretion in oxLDL-stimulated macrophages rescued normal endothelial function.